Molecular Imaging and Therapy
The current invention pertains to agents for use in molecular imaging and therapy. Molecular imaging uses exogenous contrast agents administered to the patient, whose presence within the body can be detected using medical imaging techniques. Molecular contrast agents comprise a medical imaging contrast agent bound to a targeting ligand. The contrast agent is the contrast-producing substance that is detectible by medical imaging, while the ligand is responsible for binding of the agent to the intended molecular target within the body.
Many potential targets for molecular imaging have been discovered in recent years. Similarly, the field boasts a wealth of medical imaging contrast agents able to be detected by all established medical imaging techniques. However, a key challenge in the field remains identification of suitable ligands for a given target. Specificity for the intended target is of paramount importance. Affinity and on-rate of binding are important for intravascular contrast agents. In addition, the suitability of a given ligand for conjugation to a contrast agent is important. The ability to produce a ligand at high purity and high volume is also critical. Finally, the absence of immunogenicity is desired. For each of these reasons, targeting ligands derived from or based on endogenously occurring human proteins can offer significant benefits.
Selectins as Molecular Markers of Disease
Selectins are carbohydrate-binding transmembrane molecules expressed on endothelial cells, platelets and leukocytes. They have an N-terminal C-type lectin domain. Two members of the selectin family have particular relevance in the context of molecular imaging: P-selectin and E-selectin. Up-regulation or expression of P- and E-selectin on the vascular endothelium is known to occur under conditions of inflammation, while the presence of endothelial selectins under resting conditions is generally low to nil. Disease states in which selectins are useful molecular imaging targets include post-ischemic injury, acute coronary syndrome, arthritis, inflammatory bowel disease including ileitis and colitis, atherosclerosis, myocarditis, thrombosis and multiple sclerosis. Selectin molecular imaging may be useful to deliniate and identify tissues in which selectin expression occurs under normal conditions, such as the skin microvasculature.
Up-regulation of P-selectin (CD62P) is known to occur very rapidly (within minutes), making P-selectin a potential marker of early stages of inflammatory disease. P-selectin is also found on the surface of activated platelets, making it a marker of thrombosis.
E-selectin (CD62E) is also expressed on inflamed vasculature, although generally later in the inflammatory response than P-selectin. E-selectin is thus a useful marker of inflammation at later stages of the disease.
Robust detection of inflammatory disease may be accomplished through the use of a molecular imaging probe able to detect both P- and E-selectin. Thus, it is desirable in many cases for an imaging agent intended for detecting inflammation to exhibit reactivity for both P- and E-selectins.
Differential expression of P- and E-selectin is found in some disease states. A molecular imaging agent able to bind either P- or E-selectin may be broadly useful for imaging a wide variety of disease states.
There is prior art pertaining to the use of selectins as a molecular imaging target. Use of selectins as imaging targets is contemplated, for example, in U.S. Pat. No. 6,139,819 (Unger et al); U.S. Pat. No. 6,680,047 (Klaveness et al) and U.S. Pat. No. 6,254,852 (Glajch et al). Lindner (Circulation, 2001) conjugated a rat-anti-mouse P-selectin antibody to a microbubble contrast agent and demonstrated successful detection of post-ischemic kidney injury in mice. Other studies have demonstrated use of selectins as imaging targets for detection of inflammatory bowel disease (Deshpande et al, Radiology 2012), post-ischemic myocardial injury (Villanueva et al, Circulation 2007; Leng et al, 2014), and thrombosis (Guenther et al, Invest Radiol 2010). P-selectin has also been used as a target for targeted drug delivery, for example Xie et al (J. Am. Coll. Cardiol. 2012).
Selectin-Binding Ligands
Several classes of molecules have been contemplated as ligands for imaging of selectins. Antibodies, including Fab fragments, Fv fragments, single chain molecules, and humanized anti-human antibodies, have been contemplated in this regard.
Synthetic (not naturally occurring) peptides with affinity for selectins have been described, for example in U.S. Pat. No. 5,643,873 (Barrett et al), U.S. Pat. No. 7,470,658 (Fukuda et al). Several of these have been contemplated for molecular imaging: Funovics et al (Neoplasia 2005), Zinn et al (Arthritis and Rheumatism, 1999), Gratz et al (Nuc. Ned. Comm, 2001) and Jinn et al (Cont. Med. Mol. Imaging, 2009).
Another class of ligands comprises structures based on the known endogenous selectin ligands. PSGL-1 is the most well characterized selectin ligand. PSGL-1 is a cell surface protein that exists as a dimer and contains extensive O-linked glycosylation. Various forms of PSGL-1 have been contemplated as ligands for selectin imaging, for example Davidson et al (J. Am Coll. Cardiol, 2012), and International (PCT) Publication No. WO2008131217A1 (Lindner et al). Fragments of the PSGL-1 protein have also been contemplated in this regard, for example in International (PCT) Publication No. WO2012020030A1 (Bettinger et al), Rychak et al (Mol. Pharm 2006), and Deshpande et al, (Radiology, 2012).
Various carbohydrate-based structures, some of which are components of PSGL-1, are also known to bind to selectins. This class of molecules has also been contemplated as ligands for imaging selectins, for example Villanueva et al (Circulation, 2007), Klibanov et al (Cont. Med. Mol. Imaging, 2006), and Rouzet et al (J. Nuc. Med, 2011).
TIM-1
T-cell Ig domain and mucin domain (TIM) is a family of transmembrane proteins expressed on various immune cell types, in addition to kidney and liver. Members of the TIM family are known to be involved primarily in regulation of autoimmunity (Rodriguez-Manzanet et al, 2009), although relevance in other disease processes (for example, kidney injury) has been reported (U.S. Pat. No. 6,664,385).
Of particular relevance to the current application is TIM-1 (also known as HAVCR1 (gene name), hepatitis A virus cellular receptor 1; KIM1; TIM1; HAVCR; KIM-1; TIMD1; TIMD-1; HAVCR-1). TIM-1 is a transmembrane molecule expressed on many leukocyte types, including mast cells, naïve CD4+ lymphocytes, NK cells, and some B-cells. TIM-1 is also expressed on, and shed from, renal epithelial cells in the context of kidney injury.
TIM-1 is known to bind to four molecules: TIM-1 itself, TIM-4, IGA-lambda, and phosphatidylserine. No affinity for lectins or similar molecules (including selectins), is documented in the prior art.
We have made the surprising observation that the extracellular domain of TIM-1, when conjugated to a contrast-producing agent, mediates high affinity and specific binding to selectins. As such, the extracellular domain of TIM-1 is useful for use as a ligand in the context of molecular imaging of diseases in which selectin expression is present.
The extracellular portion of TIM-1 has an N-terminal Ig-like V domain followed by a long mucin domain. The deduced amino acid sequence of human TIM-1 is shown in FIG. 1 and SEQ ID NO: 1.
Amino acids (AA) 1-20 are the signal peptide, AA 21-290 are the extracellular domain, AA 291-311 are the helical transmembrane domain and AA 312-359 are the cytoplasmic domain. Within the extracellular domain, AA 21-121 are an Ig-like V-type domain. AA 138-202 encode 11 repeats of 5 or 6 AA that encode the mucin domain. AA 203-290 encode a membrane-proximal domain. There are predicted N-linked glycosylation sites predicted at AA 65, 258, 272 and 286.
TIM-1 has been contemplated as a biomarker for kidney injury, for example U.S. Pat. No. 6,664,385 (Sanicola-Nadal et al.). However, there is no precedent for its use as a ligand for molecular imaging of selectins.